Parkin介导的线粒体自噬在酸后处理保护缺血性脑损伤中的作用及机制研究

发布时间：2018-11-28 20:30

【摘要】：缺血性脑卒中是指由于血管狭窄或闭塞造成的暂时性或永久性的脑供血不足而引起的疾病,具有高发病率、致死率、高致残率等特点。目前对于缺血性脑卒中的治疗手段依旧非常有限,唯一有效的是利用溶栓剂tPA进行溶栓处理。但由于治疗时间窗狭窄(4.5 h)和容易引起脑出血等原因限制了其临床应用。很多神经保护药物尽管在动物模型上能够减轻脑缺血损伤,但在临床上却没有明显的神经保护作用,所以亟需寻找更为安全有效的治疗手段。缺血后处理被认为是一种新型的治疗脑缺血的策略,其定义为在缺血再灌早期给予一个反复夹闭血管的亚致死刺激来激活内源性保护通路。但缺血后处理操作复杂而且处理时间窗窄,因此探寻更安全有效的后处理方法就尤为必要。除了缺糖和低氧,组织酸化也是缺血中一个重要的因素。在缺血过程中,组织的pH会从7.0下降到6.6。有研究发现在离体心脏模型中,酸化再灌可以模拟缺血后处理带来的心脏保护作用。我们课题组的前期结果发现,酸预处理可以显著的减少缺血脑损伤。由于缺血的不可预知性,研究酸后处理的神经保护作用就尤为重要。本研究旨在研究酸后处理的保护作用模式及其机制。在体缺血模型利用成年的C57BL/6J小鼠进行大脑中动脉栓塞60 min,在再灌5,50,100 min分别吸入10%,20%或30% CO2 5或10 min进行酸后处理。而离体缺血模型则在原代培养的脑片及皮质纹状体切片上进行氧糖剥夺处理,再灌不同时间后用C02平衡过的培养液孵育进行酸后处理。我们的结果发现在再灌5 min后吸入5 min浓度为10%、20%的CO2可以显著的减少缺血引起的脑梗死,并且在再灌50 min后保护作用仍然存在,提示酸后处理有更宽的治疗时间窗。而利用NaHCO3来逆转脑内的酸后可以取消酸后处理和缺血后处理的神经保护作用。并且无论是在体还是离体模型中酸后处理都可以激活线粒体自噬。并且利用3-MA, Mdivi-1和ATG7小干扰来抑制线粒体自噬都可以逆转酸后处理的保护作用。进一步我们发现酸后处理可以促进Parkin招募到线粒体上,并且敲除Parkin可以取消酸后处理带来的神经保护作用。另外我们发现酸后处理激活的线粒体自噬可以延长溶栓有效的时间窗；增加线粒体自噬可以延长酸后处理的时间窗。综上所述,在再灌早期给于一个短暂温和的酸处理可以通过激活Parkin依赖的线粒体自噬减轻缺血性脑损伤,并且其治疗时间窗优于传统的缺血后处理,因此酸后处理是一种更加有前景的内源性保护策略,线粒体自噬也是脑缺血治疗的可调控靶点。
[Abstract]:Ischemic stroke is a kind of disease caused by temporary or permanent cerebral insufficiency caused by stenosis or occlusion of blood vessels. It has the characteristics of high morbidity, mortality, high disability rate and so on. The current treatment for ischemic stroke is still very limited, the only effective thrombolytic agent tPA thrombolytic treatment. However, its clinical application was limited by treatment time window stenosis (4.5 h) and intracerebral hemorrhage. Although many neuroprotective drugs can attenuate cerebral ischemia injury in animal models, they have no obvious neuroprotective effect in clinic, so it is urgent to find more safe and effective treatment methods. Ischemic postprocessing is a novel strategy for the treatment of cerebral ischemia, which is defined as the activation of endogenous protective pathway by sublethal stimulation of repeatedly clipping blood vessels in the early stage of ischemia-reperfusion. However, the operation of ischemic post-processing is complicated and the processing time window is narrow, so it is necessary to explore more safe and effective post-processing methods. In addition to sugar deficiency and hypoxia, tissue acidification is also an important factor in ischemia. During ischemia, tissue pH decreased from 7. 0 to 6. 6. It has been found that in isolated heart model, acidified reperfusion can simulate the cardioprotective effect of ischemic post-treatment. Early results of our group showed that acid preconditioning could significantly reduce ischemic brain injury. Due to the unpredictable nature of ischemia, it is particularly important to study the neuroprotective effect of acid postprocessing. The purpose of this study was to study the protective mode and mechanism of acid post-treatment. Adult C57BL/6J mice were used for 60 min, of middle cerebral artery embolization (MCAE) in vivo ischemia model. The rats were treated with 10 ~ 20% or 30% CO2 5 or 10 min after reperfusion for 50 ~ 100 min. In vitro ischemia model was treated with oxygen and glucose deprivation in primary cultured brain slices and cortical striatum slices and then incubated with C02 balanced culture medium for acid post treatment after different time of reperfusion. Our results showed that after 5 min of reperfusion, inhaled 5 min (10 ~ 20%) of CO2 could significantly reduce the cerebral infarction induced by ischemia, and the protective effect was still present after reperfusion for 50 min, suggesting that there was a wider therapeutic time window for acid post-treatment. Using NaHCO3 to reverse the neuroprotective effects of acid-treated and ischemic post-processing in the brain. Both in vitro and in vitro, acid postprocessing can activate mitochondrial autophagy. The inhibition of mitochondrial autophagy by small interference of 3-MA, Mdivi-1 and ATG7 could reverse the protective effect of acid post-treatment. Furthermore, we found that acid post-treatment could promote Parkin recruitment into mitochondria, and knockout of Parkin could cancel the neuroprotective effect of acid post-treatment. In addition, we found that activated mitochondrial autophagy after acid treatment can prolong the effective time window of thrombolysis, and increase mitochondrial autophagy can prolong the time window of acid post treatment. In conclusion, given a transient mild acid treatment at the early stage of reperfusion can reduce ischemic brain damage by activating Parkin dependent mitochondrial autophagy, and its therapeutic time window is superior to that of conventional ischemic postconditioning. Therefore, acid aftertreatment is a more promising endogenous protection strategy, mitochondrial autophagy is also a regulatory target for cerebral ischemia therapy.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.3

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